Technical Abstract:
The reliability of microarray data is critical for obtaining meaningful biological insights from genomic expression analysis. Quality control of microarray experiments is an important element for microarray data handling and variation estimate of microarray experiments. In generating fungal and bacterial DNA oligo microarrays for functional genomic studies, we developed exogenous nucleic acid controls using ACTB (beta-actin), B2M (beta-2-microglobulin), and PGK1 (phosphoglycerate kinase 1) from bovine (Bos taurus), and Cab (photo system I chlorophyll ab binding protein), MSG (major latex protein), and RBS1 (ribulose bisphosphate carboxylase small chain 1 precursor) from soybeans (Glycine max). A unique 70-mer DNA oligo for each gene was synthesized with the 5' amine-modified and printed on to covalently binding slides incorporated with a high density microbial DNA oligo microarray, such as the yeast genome for Saccharomyces cerevisiae and a new Pseudomonas genome for Pseudomonas fluorescens Pf-5. Multiple control gene spots are evenly printed and distributed on the entire microarray slide. In addition, six sets of the control genes and two negative controls are printed on top of each microarray for prescanning to adjust PMT Gain prior to scanning the entire array. The mRNA for each gene was transcribed in vitro and quantified through spectrophotometry and gel electrophoresis. Two sets of mRNA mix, CtrlBt and CtrlGm, were prepared, and each consisted of three genes originating from B. taurus and G. max, respectively. Each mix consisted of a defined high, medium, and low concentration from each of the three genes of the same species origin. CtrlBt and/or CtrlGm are applied as spiking controls along with a sample labeling reaction to be transcribed into cDNA and incorporated with a labeling dye. Fluorescence intensities of the controls are used to examine the linearity of the array signal intensity, to balance the Cy-dye intensities, to evaluate the array sensitivity, to measure the expression consistency, to estimate print quality, and to evaluate hybridization consistency. These spiking controls can be used as a positive control and reference for data acquisition and normalization. The coefficient of variance of the microarray can be consistently calculated using the control genes. For DNA oligo microarrays of general bacterial and fungi, either set of controls or both can be applied. For an extended use of microarrays of animal or animal-associated microorganisms, CtrlGm is recommended; while for those of plant or plant-associated microorganisms, CtrlBt can be applied.